Fluorescence polarization is a known powerful method for the rapid and homogeneous analysis of molecular interactions in biological and chemical systems.
The principles of fluorescence polarization are based on the excitation of a fluorescent molecule, the fluorophore, with polarized light. This results in the emission of photons in the plane which is parallel and perpendicular to the excitation plane and yields information about the local environment of the fluorophore.
The rotation of fluorophores in solution can be observed by measuring the rotation of the plane of polarization of the light that was originally beamed in.
The polarization value for solved randomly oriented molecules is between 0 and 0.5 depending on the rotational speed of the molecules during the fluorescence lifetime of the excited state. If the rotation is rapid in comparison with the fluorescence lifetime, the polarization of the emitted radiation is almost zero. If, in contrast, the rotation is slow compared to the fluorescence lifetime, the polarization in the emitted light is high. Small molecules rotate rapidly and therefore have a smaller polarization value. Conversely larger molecules have a higher polarization value due to their slow rotation.
One of the advantage of fluorescence polarization is that variations in the intensity in the optical system and in the sample solution are automatically eliminated by relating the various polarized emission components to one another. Hence errors caused by the optical system or by inconsistent samples e.g. samples that absorb to different extents, do not usually occur with this method.
The observed rotation depends on the rotation relaxation time and is only influenced by the temperature, the viscosity and the molecular weight of the fluorophore. Thus fluorescence polarization is a suitable method for measuring these parameters and in particular changes in these parameters.
The use of static and dynamic fluorescence polarization to diagnostically detect complexation reactions is know in prior art. In this method an antigen that is labelled with a fluorophore and is present in solution is reacted with an antibody which is present in a diluted serum sample in order to form an immune complex. The immune complex is then detected by the change in fluorescence polarization.
It is also known that fluorescence polarization is used as an analytical tool for immunoassays e.g. to detect serum proteins, antibodies and hormones, to detect toxins in seeds and for drug detection.
In such tests one generally uses fluorescently-labelled antigens or antibodies whose polarization is measured. Their reaction with a specific antibody or antigen results in an increase in fluorescence polarization i.e. after an antigen-antibody complex has been formed which increases the effective molecular weight.
The application of fluorescence polarization as a tool for investigating polyelectrolyte complexes and hydrogels is also known. In this method the fluorophore is covalently linked with the polymer to be examined.
A disadvantage of the known diagnostic methods in which fluorescence polarization is measured, is that a laborious optimization of the label i.e. of the fluorophore and of its binding to the antigen or antibody is required to prevent an impairment of the actual complex formation. Furthermore labels that are to be used to detect changes in molecular weight by fluorescence polarization have to be bound covalently to the labelled molecule since even small relative movements reduce the fluorescence polarization and thus the effective signal.
The fluorescence resonance energy transfer (FRET) method is another analytical method in which fluorophores are used. In the FRET method photon energy is transferred in a non-radiative manner from an excited fluorophore (the donor) to another fluorophore (the acceptor) when the distance between the two of them is no more than 1-10 nm. This energy transfer occurs non-radiatively, essentially by a dipole/dipole interaction. The FRET method can also be used for example to detect molecular interactions between two protein partners or structural changes within a molecule.
However, a disadvantage of the FRET method is that only certain fluorophore pairs are suitable for the FRET method since, as apart from other prerequisites such as dipole orientation and adequate fluorescence lifetime, the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor. Two labels are necessary which is why the FRET method is even more complicated than fluorescence polarization.
Various methods are known from the prior art for detecting glucose. One such method is the detection of glucose by means of the complexation reaction concanavalin A (ConA)-dextran. Glucose interferes with the complex formation which results in a change in viscosity. Hence the complex formation and a change thereof and thus the concentration of glucose can be measured by means of viscosimetry. A disadvantage of this method for detecting glucose is that the viscosimetric method is technically very complicated.
Another method for determining glucose is based on the FRET method. In this method, Concanavalin A and dextran can both be labelled with a fluorophore. The label results in an effective energy transfer from the first to the second fluorophore (FRET) in the complex during complex formation. The addition of glucose leads to a dissolution of the complex which decreases the intensity of the radiation from the second fluorophore. A major disadvantage of detecting glucose using the FRET method is that concanavalin A as well as dextran have to be labelled with a fluorophore. Moreover, the corresponding fluorophores must have suitable fluorescence properties. Reabsorption of the radiation emitted from the first fluorophore by the second fluorophore also reduces the effective signal since it also occurs without complex formation.
In addition to the methods described above, several methods methods are known for continuously monitoring glucose in vivo and in vitro> For example microdialysis and enzymatic detection outside the body; viscosimetry using concanavalin A/dextran (GlucOnline®); electrochemical sensors employing an enzymatic conversion of the glucose in the body (Minimed®); long-term sensors which for example operate according to the FRET method on the basis of labelled concanavalin A and dextran, have all been used to detect glucose.
However, no method or system is known in which an in vivo glucose monitoring for determining the glucose concentration or an in vitro determination of the glucose concentration can be carried out by means of fluorescence polarization.
Therefore, an object of the invention is to overcome the disadvantages of the prior art and to provide a simple and accurate method for in vivo glucose monitoring by means of fluorescence polarization as well as a system which can be used in a method for in vivo glucose monitoring.
These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is definitely by the recitations therein and not by the specific discussion of the features and advantages set forth in the present description.